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Vitamins B₆, B₁₂, and Folate: Association with Plasma Total Homocysteine and Risk of Coronary Atherosclerosis

Department of Food Technology and Nutrition Sciences (P.W.S., P.V., F.J.K.), Division of Human Nutrition and Epidemiology, Wageningen Agricultural University, Wageningen, THE NETHERLANDS
Netherlands Institute of Health Sciences (P.W.S.), Erasmus University Medical School, Rotterdam, THE NETHERLANDS
Institute of Human Nutrition (P.W.S.), Columbia University, New York, New York

Address reprint requests to: Petra Verhoef, PhD, Department of Food Technology and Nutrition, Division of Human Nutrition and Epidemiology, Wageningen Agricultural University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands

	ABSTRACT

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Objectives: To investigate the association of status of vitamins B₆, B₁₂ and folate with plasma fasting total homocysteine (tHcy) and with risk of coronary atherosclerosis; and to establish whether associations between vitamins and risk of coronary atherosclerosis are mediated by tHcy.

Methods: The study population consisted of 131 patients with angiographically-defined severe coronary atherosclerosis and 88 referents with no or minor coronary stenosis. Previous analyses in this study population have shown that fasting tHcy is an independent risk factor for coronary atherosclerosis. In the present analyses, using multiple linear regression, we estimated differences in tHcy concentrations between subjects in the lowest and highest quartiles of concentrations of each of the vitamins, adjusting for age, gender, total:HDL cholesterol ratio, smoking habits, alcohol intake, blood pressure, serum creatinine, body mass index and the two other vitamins. We used logistic regression analysis conditional on the set of potential confounders described above to study the association between vitamin concentration and risk of coronary atherosclerosis. By comparing these estimated odds ratios (ORs) with those that were additionally adjusted for fasting tHcy, we determined whether the vitamins exerted their effects on disease risk via homocysteine metabolism.

Results: Cases who were in the upper quartile of serum vitamin B₁₂ and erythrocyte folate concentrations showed statistically significantly lower tHcy concentrations (-4.00 and -4.71 µmol/L, respectively) than those in the lowest quartile. Referents in the upper quartile of plasma B₆ showed significantly lower tHcy concentrations (-2.36 µmol/L) than referents in the lowest quartile. Subjects in the lowest quartile of vitamin B₁₂ concentration had higher risk of coronary atherosclerosis (OR: 2.91; 95% CI: 1.10, 7.71) compared to those in the highest quartile. The ORs and 95% CIs for low B₆ and low folate were 0.86 (95% CI: 0.33, 2.22) and 0.58 (95% CI: 0.23, 1.48), respectively. Additional adjustment for fasting tHcy weakened associations, although data indicated that low vitamin B₁₂ concentration is a risk factor for coronary atherosclerosis, independently of tHcy.

Conclusion: The presently accepted view that vitamin B₆ mainly affects tHcy after methionine loading, and not fasting tHcy, is contradicted by our findings in referents. Low vitamin B₁₂ concentrations were associated with an increased risk of coronary atherosclerosis, partly independently of tHcy. Although low folate status was a strong determinant of elevated tHcy concentrations, it was not associated with increased risk of coronary atherosclerosis.

Key words: vitamin B₆, vitamin B₁₂, folate, plasma total homocysteine, coronary atherosclerosis, methylenetetrahydrofolate reductase (MTHFR)

	INTRODUCTION

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Though increasingly more evidence supports the hypothesis that elevated total homocysteine (tHcy) is an independent risk factor for vascular disease of the coronary, cerebral and peripheral vessels [1–7], the precise role of B₆ vitamins in the determination of either plasma tHcy or risk of vascular disease remains a point of contention. While some studies support the hypothesis that inadequate vitamin levels can increase cardiovascular disease risk [8–13], other investigations have found little or no evidence for such a relationship [14–16], particularly with regard to vitamin B₆. The precise relationship between the B vitamins and tHcy, and its associated effects on disease risk, have also remained undefined, with some studies finding no difference in concentrations of B₆, B₁₂ and/or folate between patients and referents despite higher tHcy in cases [11,17,18].

Folate and vitamins B₆ and B₁₂ are essential components in the metabolism of tHcy, which occurs through remethylation to methionine or transsulfuration to cysteine. The enzyme methylenetetrahydrofolate reductase (MTHFR) is responsible for the reduction of 5,10-methylene-THF to 5-methyl-THF, the required substrate in the remethylation process where B-12 acts as a cofactor. Transsulfuration of tHcy to cysteine relies on a vitamin B₆-dependent enzyme, cystathionine ß-synthase [19,20]. Inherently, reduced enzyme activity in a number of places may lead to an increase in tHcy levels which in turn may increase risk of coronary atherosclerosis. Recently, a 677 C to T mutation in the MTHFR gene has been described [21] rendering the enzyme thermolabile, and thereby leading to elevation of tHcy levels (mainly fasting levels) in subjects homozygous for the mutant gene, especially in those with low folate status [22–24].

Previous analyses in our study population have shown that fasting tHcy is an independent risk factor for coronary atherosclerosis [4]. The present study attempted to clarify the association between the B vitamins and risk of coronary atherosclerosis. We also investigated how plasma tHcy varied with concentrations of vitamins B₆, B₁₂ and folate, and studied the association between the B vitamins and risk of coronary atherosclerosis after correction for tHcy, in order to address the hypothesis that vitamins affect risk of atherosclerosis through the homocysteine pathway. Furthermore, we examined the association between folate erythrocyte concentration and risk of atherosclerosis by MTHFR genotype subgroups since mutation of the MTHFR gene may lead to a higher folate requirement in tHcy regulation [22,23], which was also observed in previous analyses in our study population [24].

	MATERIALS AND METHODS

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Study Population
A case-referent study was conducted from June 1992 to June 1994. Cases of coronary atherosclerosis and a referent group were selected from patients aged 25 to 65 years undergoing coronary angiography at the Zuiderziekenhuis Hospital in Rotterdam, The Netherlands. Inclusion criteria for all study participants required that they had no history of diabetes, renal, hepatic or gastrointestinal disease, cancer, alcohol or drug abuse, or psychiatric illness.

At angiography, the extent of occlusion of the coronary vessels was determined using standard catheterization techniques. A team of cardiologists reviewed the projections and prepared reports which were then used by a trained research nurse to select potential cases and coronary referents. Those persons with 90% occlusion in one artery and at least 40% occlusion in another were defined as cases of severe atherosclerosis. Coronary referents could have no more than 50% occlusion in one coronary artery.

During the 2-year study period, 2,659 patients underwent coronary angiography. Of these, 2,292 were not selected for participation in the study due to age over 65 years, intermediate coronary occlusion and/or failure to meet other inclusion criteria. There were 369 eligible subjects who were subsequently invited to participate. Nearly all of these persons (95.6%) could be reached, and of those, 222 (63%) were willing to participate, yielding 131 cases and 91 coronary referents. A second evaluation of angiography results deemed three of the coronary referents ineligible to participate due to excessive coronary narrowing which was not sufficient to define them as cases, however.

In the selected study population, 77.1% of the cases had at least 70% occlusion in a second vessel. Furthermore, the majority of coronary referents did not show substantial narrowing in any of the three arteries; in fact, only 5.7% reached the limit of having 50% stenosis in a single vessel. The marked contrast between cases and coronary referents reduced the possibility of disease misclassification. Histories of myocardial infarction (MI) were found in half of the selected cases (n=67) and 7% of the selected coronary referents (n=6). In the coronary referents, the myocardial infarctions were due to coronary spasms or other non-atherosclerotic causes. Because coronary atherosclerosis was the endpoint of interest, referents with a history of MI were not excluded from the study.

The study protocol was approved by the medical ethics committee. All participants gave their written informed consent.

Blood Sampling and Examination
Venous blood samples were obtained from all subjects between 8:30 and 9:30 a.m., after a 10 to 12 hour fast. Height and weight (without shoes and heavy clothing) were also measured. Subjects were interviewed about current and past smoking habits, alcohol consumption patterns and use of medication.

For measurement of whole blood folate, 200 µl of blood sampled in tubes containing ethylene diamine tetraacetic acid (EDTA) was mixed with 4 ml (1:20) freshly prepared 1% (w/v) ascorbic acid solution. The rest of the EDTA blood was placed in the dark on immediately, and centrifuged at 4°C within an hour. The serum and plasma samples were stored at -80°C for a maximum of 6 months before determination of concentrations of total homocysteine, creatinine, pyridoxal 5'-phosphate (PLP) as a metabolite of vitamin B₆ and plasma vitamin B₁₂ as cobalamin took place.

Biochemical Analyses
Plasma total fasting homocysteine (tHcy) which refers to the sum of protein-bound, free-oxidized and reduced species of homocysteine in plasma, was determined by an automated assay based on precolumn derivatization with monobromobimane, followed by high pressure liquid chromatography (HPLC) separation and fluorescence detection [25]. The assay was performed at the Department of Clinical Biology, Division of Pharmacology, University of Bergen, Norway. All estimations were performed in duplicate and the coefficient of variation for these analyses was 3%.

All other determinations were performed at the laboratory of MIMELAB-AB, Söråker, Sweden. To estimate plasma PLP, enzymatic photometry with HPLC separation was used. Radioimmunoassay was used to determine whole blood folate and vitamin B₁₂ as well as serum plasma vitamin B₁₂. Folate concentration has been expressed per haematocrit, that is, as erythrocyte folate. Serum creatinine, total cholesterol and HDL cholesterol (after precipitation of LDL and VLDL) were determined with enzymatic photometry.

For MTHFR genotyping, DNA was obtained from the buffy coat of EDTA blood. The mutation involves a C to T alteration at nucleotide 677 which converts an alanine to a valine residue. This creates a HinFI site which can be used for mutation analysis as has been described in detail elsewhere [21].

Data Analysis
Vitamins and Coronary Atherosclerosis.
Cardiovascular risk factors were compared for the 131 cases of coronary atherosclerosis and the 88 referents. All vitamin and tHcy measures were positively skewed, so that logarithmic transformations were used to normalize their distributions. Differences between cases and referents were tested for statistical significance with Student t-test (continuous variables) or Chi-square test (categorical variables).

Odds ratios (ORs) for coronary atherosclerosis and their 95% confidence intervals (CIs) were calculated from the parameter estimates and standard errors from a logistic regression model for quartiles of vitamin concentrations defined according to the referent distribution with the highest quartile as the reference. Age, gender, heavy smoking years, blood pressure, total:HDL cholesterol ratio, alcohol intake, body mass index, serum creatinine and levels of the other two B vitamins were considered as potential covariates through stratified analysis. ORs were ultimately calculated conditional on this entire set of potential confounders, and these estimates were then compared to ORs which were additionally adjusted for tHcy to evaluate any tHcy-independent effect of the vitamins.

We also examined the association between folate concentration and risk of coronary atherosclerosis according to MTHFR subgroups since mutation of the MTHFR gene may lead to a higher folate requirement in tHcy regulation [22–24]. ORs were computed with adjustment for age and gender. Changes in these OR estimates after further adjustment for fasting tHcy were then evaluated.

Vitamins and Plasma Total Homocysteine.
We studied the associations between fasting tHcy and vitamins B₆, B₁₂ and folate in two ways. Firstly, using multiple linear regression, we examined how tHcy varied according to vitamin concentrations in cases and referents separately after adjustment for age, gender, heavy smoking years, blood pressure, total/HDL cholesterol ratio, alcohol intake, body mass index, serum creatinine and levels of the other two B vitamins. Adjusted differences in tHcy concentrations were calculated for those with high vitamin concentrations (75th percentile) compared to those with low vitamin concentrations (25th percentile).

Secondly, among groups with elevated and non-elevated homocysteine levels (elevated levels were defined at the 75th percentile of the referent distribution), we compared the prevalence of persons with low vitamin status (defined as 25th percentile of any of the B vitamins). For all analyses, p-values were two-sided.

	RESULTS

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Vitamins and Coronary Atherosclerosis
Table 1 shows the baseline characteristics and vitamin concentrations of cases and referents. There were significantly more males among cases than referents. Cases were significantly older, had a significantly higher total/HDL cholesterol ratio, had significantly higher serum creatinine concentrations and were significantly more often hypertensive. Although the percentage of current smokers did not greatly differ, cases smoked heavily for a significantly longer period of time. Other differences in coronary risk factors were not statistically significant. After adjustment for age and gender, cases had slightly lower plasma concentrations of PLP and vitamin B₁₂, but higher erythrocyte folate than referents. However, none of these differences were statistically significant.

When we further adjusted the models for fasting tHcy to examine the independent effects of the B vitamins on risk of coronary atherosclerosis, the OR estimate for vitamin B₁₂ decreased from 2.91 to 2.23 (95% CI:0.81, 6.09). The ORs of disease for the lowest quartiles of vitamin B₆ and folate concentrations changed slightly, but none of these estimates was statistically significant.

The relationship between folate and coronary atherosclerosis was further explored in subgroup analysis according to MTHFR genotype. There were 13 (9.9%) cases and 10 (11.4%) referents who were homozygous for the mutant gene (+/+); 59 (45.0%) cases and 43 (49.4%) referents were heterozygous (+/-); and 59 (45.0%) cases and 34 (39.1%) referents were homozygous normal (-/-). The ORs for atherosclerosis for subjects in the lowest quartile of erythrocyte folate compared to those in the upper three quartiles of the referent distribution, adjusted for age and gender, were 0.24 (0.07, 0.80), 0.75 (0.26, 2.18) and 8.95 (0.43, 188) for -/-, +/- and +/+ subjects, respectively. After additional adjustment for fasting tHcy, the respective ORs changed to 0.16 (0.042,0.63), 0.67 (0.22,2.01) and 6.61 (0.22, 196).

Vitamins and Plasma Total Homocysteine
Hyperhomocysteinemia (defined as tHcy levels above the 75th percentile of referents (13.3 µmol/L)) occurred in 52 (39.7%) of the cases compared to 22 (25.0%) of the referents (Chi-square test: p=0.024). Significantly more persons with a vulnerable vitamin profile (defined as 25th percentile of any of the B vitamins) were among those subjects with elevated tHcy in comparison to those with normal levels of tHcy (72% vs. 50%) (Chi-square test: p=0.002).

Plasma concentration of tHcy was found to be inversely associated with vitamins B₆, B₁₂ and folate in multivariate adjusted regression analyses (Table 3). In cases, vitamins B₁₂ and folate were significantly associated with tHcy concentrations while in referents, only vitamin B₆ was found to be significantly associated with concentrations of tHcy.

	DISCUSSION

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The observation for vitamins and tHcy in cases supports the idea that vitamins B₁₂ and folate are associated with fasting tHcy through their involvement in homocysteine remethylation [19]. However, tHcy concentrations in our study referents were only significantly associated with concentrations of vitamin B₆, a finding which has in fact been reported in some early animal studies [26–28]. Generally, low concentrations of vitamin B₆ are associated with increased tHcy after methionine-loading, reflecting reduced transsulfuration. The observed association with fasting tHcy may be related to the role of vitamin B₆ as a co-enzyme for the formation of methylene-THF from THF [20]. More studies are needed to investigate the precise association of vitamin B₆ with fasting tHcy and further, the possibility that a subject’s disease state somehow influences the biological pathways in which this vitamin is involved.

Despite its clear association with tHcy in referents, vitamin B₆ was not clearly related to risk of atherosclerosis as seen by the OR estimates over quartiles of the distribution given in Table 2. Furthermore, although folate and vitamin B₁₂ were significant predictors of tHcy in cases, and tHcy and coronary atherosclerosis were positively associated as described in a previous report [4], only lowest levels of vitamin B₁₂, but not of folate, were significantly associated with increased risk of atherosclerosis. The fact that the OR estimate for low vitamin B₁₂ was only modestly reduced after adjustment for fasting tHcy further implied that the relationship between vitamin B₁₂ and risk of atherosclerosis was only partially mediated by effects on plasma tHcy.

Other retrospective case-referent studies [11,17] and a recent prospectively-designed one [18] have also demonstrated inverse associations between B vitamins and tHcy and positive associations between tHcy and CVD risk, despite equivalent or even higher levels of vitamins in cases. The latter study [18], for example, showed only slightly, nonsignificantly lower plasma concentrations of folate and PLP in patients of myocardial infarction compared to their age-matched referents in spite of higher fasting tHcy concentrations in cases. Similarly, although Robinson et al [11] and Dalery et al [17] demonstrated higher tHcy concentrations in cases of coronary disease than in referents, neither study showed lower folate concentrations in cases. On the other hand, two other prospective studies [9,10] have shown that low folate status was associated with increased risk of ischemic stroke and coronary heart disease. These studies had no information on tHcy concentrations.

Our observation that cases did not have lower levels of folate, despite higher tHcy, may imply that these persons need more folate to keep tHcy at a normal level, possibly due to a hereditary defect in tHcy metabolism (e.g., thermolabile MTHFR). However, the prevalence of the MTHFR mutation was not greater in cases than in referents (9.9% vs. 11.4%) which suggests that other factors, e.g., medication, may have increased folate demand. We found an OR for atherosclerosis of 8.95 (95% CI: 0.43, 188) for subjects in the lowest quartile of folate compared to the three upper quartiles among persons homozygous for the mutant gene which suggests that low folate status may only be a risk factor for coronary atherosclerosis in a population with a high percentage of persons with the +/+ genotype. This OR estimate, however, must be viewed skeptically in light of the wide CI. Adjustment here for tHcy revealed only a partial attenuation of the association, suggesting that although folate may exert part of its effect on risk of atherosclerosis via its effect on tHcy, the vitamin may also be related to risk of coronary atherosclerosis through other mechanisms.

The higher folate concentrations found among cases may be due to changes in lifestyle after disease diagnosis. In fact, we observed that 39.7% of cases changed their dietary habits in comparison to only 22.0% of referents (p=0.002). Though the main thrust of dietary change was to lower fat intake, increased consumption of fruits and vegetables may have also taken place. This may have attenuated the associations between vitamins and coronary atherosclerosis, but probably not the vitamin-tHcy association, since tHcy levels reflect recent dietary habits. This could partly explain the discrepancy in findings between the vitamin-tHcy and tHcy-coronary atherosclerosis relationships.

Another consideration for that discrepancy may be that plasma and serum concentrations of vitamins do not always reflect tissue deficiency of vitamins [29]. It may be more important to evaluate risk of coronary disease based on elevated metabolite levels rather than sub-optimal plasma and serum vitamin concentrations, since the former may more accurately reflect lower tissue concentrations [30] as one prospective, multi-center, double-blind controlled study [31] has found. By randomizing elderly persons with elevated metabolite concentrations, including tHcy, but normal serum vitamin concentrations, to treatment with either an intramuscular B vitamin supplement or placebo, Naurath et al showed that concentrations of tHcy and other metabolites of the group receiving vitamins were significantly reduced in comparison to the placebo group.

Certainly, choice of the proper referent group is of major consideration for any case-referent study. This study used coronary referents as the group of comparison which allowed us to be certain of the extent of atherosclerosis. Using coronary referents also ensured that referents selected were from the catchment population from which the cases were derived. However, a disadvantage of using such referents is that the group is inherently a high-risk one with a clinical profile severe enough to mandate catheterization. Coronary referents may therefore represent a group with other abnormalities which may somehow influence the determinant-disease relationship.

In conclusion, in line with other studies, we have shown that B vitamins are significantly associated with plasma tHcy which, at elevated concentrations, increases risk of coronary heart disease. Low B₁₂ concentrations were significantly associated with increased risk of coronary atherosclerosis, partly independent of tHcy. Data suggested that low folate status was related to a higher risk of atherosclerosis in subjects homozygous for the MTHFR mutation only. The conflicting results for the vitamin-tHcy relationship on the one hand and the vitamin-coronary atherosclerosis relationship on the other hand are of the same nature as those often encountered in other observational studies, particularly in case-referent studies. These results may be explained by biases e.g., changes in lifestyle among cases or the effects of medication, which may affect case-referent studies, or they may indicate that tissue concentrations of vitamins rather than serum or plasma concentrations may be a more reliable parameter in the evaluation of vitamin status. Prospective studies investigating the relationship between intake of the vitamins and risk of cardiovascular disease will be useful in clarifying this issue, provided that usual dietary intake can be adequately measured.

	ACKNOWLEDGMENTS

 
The study was supported by a grant from the Netherlands Organization for Scientific Research. The authors would like to express their gratitude to Drs. A.A.A. Bak, S.C. Balduw, G.J. van Beek, M.P. Freericks, F.M.A. Harms, R. van Mechelen, W.M. Muijs van de Moer and R. Wardeh for their support in selecting the participants. We are grateful to Annelies Legters, the research assistant. We thank Drs. Dick Kruyssen, Jacqueline Witteman, Evert Schouten, Diederick Grobbee, Henk Blom, Leo Kluijtmans, Helga Refsum and Per Ueland for their contribution to this study. Furthermore, we would like to thank Mariëtte Penning, Halvard Bergesen, Elfrid Blomdal, Wenche Breyholtz and coworkers from the Sticares Foundation for their assistance in this research.

Received March 1, 1997. Accepted December 1, 1997.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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